In several types of tumors, abnormal DNA methylation has been observed at the 3'-end of specific genes, called CpG islands. It has been suggested that these hypermethylated CpG islands are associated with inactivation of tumor suppressor genes. In estrogen receptor (ER)-negative breast cancer cell lines, hypermethylation of the ER Cp6 island is directly linked to the silencing of this gene. ER-negative cells also exhibited a high capacity to methyl ate DNA due to an increase in methyltransferase activity. However, it remains to be determined whether the hypermethylated ER CpG island can be observed in vivo and whether hypermethylation plays a role in silencing other gene activities. The preliminary data have led to the identification of two common hypermethylation sites in breast tumors using a modified differential display PCR method. The proposed study will further identify and characterize abnormal methylation sites from primary human breast carcinomas and study their roles in gene regulation. Hypothesis: DNA hypermethylation at CpG islands provides an alternative mechanism of genetic alterations in inactivating tumor suppressor genes from breast tumors. To conduct the study, it is important to first study ER gene methylation in vivo. Subsequently, abnormal methylation in other genes related to breast carcinogenesis will be identified. Aim 1. Determine whether the methylation status of the ER CpG island in primary breast tumors correlates with the conventional ER-binding assay. Aim 2. Identify common hypermethylation sites from primary breast tumors and determine whether these sites are associated with specific clinical parameters of the patients. Aim 3. Identify cDNAs corresponding to the common hypermethylation sites and examine the effect of hypermethylation on gene inactivation in breast cancer cells. This study will provide information important to the understanding of DNA hypermethylation in breast carcinogenesis. The characterization of its corresponding biologically relevant cDNAs will give new insights into the pathobiology and treatment of breast cancer. In addition, the identified common hypermethylation sites can be used as diagnostic "genetic fingerprints" to supplement currently used pathological markers.